A novel bivalent chromatin associates with rapid induction of camalexin biosynthesis genes in response to a pathogen signal in Arabidopsis
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Evaluation Summary:
This study proposes the identification of "bivalent chromatin" in genes associated with the biosynthesis of secondary metabolites in Arabidopsis and describes an investigation into the role of chromatin states in the regulation of the major Arabidopsis phytoalexin. Perturbation of either H3K27me3 or H3K18ac levels using mutants were used to show that there were effects on the expression of these metabolic genes. It has previously been shown that H3K27me3 and H3K18ac colocalize in the Arabidopsis genome and that genes targeted by PRC2/H3K27me3 in Arabidopsis are enriched for genes that respond to the environment and/or developmental cues. Therefore, the reported changes to the regulation of these genes in defective mutants are as expected, although the finding of this study will still be of interest to those working on pathogen-induced changes to plant metabolism.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Temporal dynamics of gene expression underpin responses to internal and environmental stimuli. In eukaryotes, regulation of gene induction includes changing chromatin states at target genes and recruiting the transcriptional machinery that includes transcription factors. As one of the most potent defense compounds in Arabidopsis thaliana , camalexin can be rapidly induced by bacterial and fungal infections. Though several transcription factors controlling camalexin biosynthesis genes have been characterized, how the rapid activation of genes in this pathway upon a pathogen signal is enabled remains unknown. By combining publicly available epigenomic data with in vivo chromatin modification mapping, we found that camalexin biosynthesis genes are marked with two epigenetic modifications with opposite effects on gene expression, trimethylation of lysine 27 of histone 3 (H3K27me3) (repression) and acetylation of lysine 18 of histone 3 (H3K18ac) (activation), to form a previously uncharacterized type of bivalent chromatin. Mutants with reduced H3K27me3 or H3K18ac suggested that both modifications were required to determine the timing of gene expression and metabolite accumulation at an early stage of the stress response. Our study indicates that the H3K27me3-H3K18ac bivalent chromatin, which we name as kairostat, plays an important role in controlling the timely induction of gene expression upon stress stimuli in plants.
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Author Response:
Evaluation Summary:
This study proposes the identification of "bivalent chromatin" in genes associated with the biosynthesis of secondary metabolites in Arabidopsis and describes an investigation into the role of chromatin states in the regulation of the major Arabidopsis phytoalexin. Perturbation of either H3K27me3 or H3K18ac levels using mutants were used to show that there were effects on the expression of these metabolic genes. It has previously been shown that H3K27me3 and H3K18ac colocalize in the Arabidopsis genome and that genes targeted by PRC2/H3K27me3 in Arabidopsis are enriched for genes that respond to the environment and/or developmental cues. Therefore, the reported changes to the regulation of these genes in defective mutants are as expected, although the finding of this study will still be of interest …
Author Response:
Evaluation Summary:
This study proposes the identification of "bivalent chromatin" in genes associated with the biosynthesis of secondary metabolites in Arabidopsis and describes an investigation into the role of chromatin states in the regulation of the major Arabidopsis phytoalexin. Perturbation of either H3K27me3 or H3K18ac levels using mutants were used to show that there were effects on the expression of these metabolic genes. It has previously been shown that H3K27me3 and H3K18ac colocalize in the Arabidopsis genome and that genes targeted by PRC2/H3K27me3 in Arabidopsis are enriched for genes that respond to the environment and/or developmental cues. Therefore, the reported changes to the regulation of these genes in defective mutants are as expected, although the finding of this study will still be of interest to those working on pathogen-induced changes to plant metabolism.
Response: In our paper, we put forward a new model for the role of a bivalent chromatin formed by H3K18ac-H3K27me3, a form that is not yet studied in the bivalent chromatin literature, on regulating the timing of a defense compound synthesis pathway in a whole organism. As far as we know, bivalent chromatin has not been associated with controlling metabolism in any system. We tested our model genetically using several mutant lines that affect both the activating and repressing marks. We show that both H3K27me3 and H3K18ac are required to maintain timely induction of camalexin genes upon a stress signal using ChIP, genetics, and biochemistry, which is a novel molecular mechanism and different from PRC2 complex mediated repression of a bivalent chromatin. This is the first time that a clear function of a bivalent chromatin has been demonstrated in vivo and at the organismal level in any system.
Reviewer #1 (Public Review):
The series of experiments to identify and examine changes in chromatin marks in response to flgg22 treatments investigate clearly defined hypotheses. They first present data showing that genes in pathways involved in specialized metabolism are more likely to be associated with both repression (H3K27me3) and activation (H3K18ac) marks than expected by chance in the genome. Using antibodies against H3K27me3 and H3K18ac in pull-down experiments, they show that H3K18ac and H3K27me3 are co-localized at the camalexin biosynthesis genes. They then show that, in response to FLG22, H3K18ac modifications increased and H3K27me3 modifications depleted. In mutant lines that have defective deposition of H3K27me3 expression of camalexin biosynthesis genes in response to FLG22 increased compared to wild type. Together, this progression of experiments provides convincing data of an association between chromatin state and changes in transcription levels. Overall, the model is compelling, though the authors should take care to ensure that the language used is reflective of the association or interplay between chromatin state and transcription factor availability.
Response: we revised the manuscript to reflect the interplay between chromatin state and transcription factor availability in Introduction: “The accessibility of target gene regions to transcription factors is determined by the dynamics of chromatin states in eukaryotic cells16. Chromatin states are controlled by epigenetic modifications that influence nucleosome accessibility17. Epigenetic modifications constitute various covalent decoration of chemical groups to histones and DNA, which are associated with promoting or repressing gene expression by altering chromatin accessibility to transcription factors. For example, trimethylation of lysine 27 of histone 3 (H3K27me3), established by the Polycomb Repressive Complex 2 (PRC2), is associated with repressing gene expression20. H3K27me3 represses gene expression by increasing chromatin condensation and limiting the recruitment of transcription factors and other components of the transcriptional machinery. Trimethylation of lysine 4 of histone 3 (H3K4me3) is marked at actively transcribed genes, which activates gene expression by promoting the recruitment of transcription initiation factors to promoters of target genes.”
What is less clear is the link between chromatin states/transcriptional expression and the abundance of the metabolic pathway products that are required to limit pathogen spread. In this manuscript, Zhao et al, test camalexin content using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following FLG22, finding an initial accumulation, followed by further increases over 6 hours. Plants with reduced H3K27me3 marks started to accumulate camalexin earlier while those with reduced H3K18ac marks accumulated camalexin later. While the altered timings in the mutant lines do support a connection between gene expression dynamics and metabolite accumulation, it does not prove that changes in transcription are wholly responsible for the differences in metabolites. Previously reported Ribo-seq experiments mRNAs from genes, including those for camalexin biosynthesis, suggesting that PTI/RTI-triggered translational regulation has a significant role in changes in expression (Xu et al 2017 Nature 545, 487-490; Yoo et al Molecular Plant 13:1 88-98). This does not need to detract from the main findings of this paper, but the changes in metabolite accumulation should be interpreted with these data in mind and discussed appropriately.
Response: We thank the reviewer for pointing this out and helping us improve the manuscript by providing a more holistic context of camalexin regulation. We revised Introduction and Discussion to include current understanding of post-transcriptional regulation on camalexin pathway and interpret the results in a more holistic view. Revisions in the Introduction: “Previous studies revealed complex transcriptional and translational control of camalexin biosynthesis genes. At the transcriptional regulation level, transcription factors from the MYB family, including MYB34, MYB51 and MYB122 promote camalexin biosynthesis in response to P. syringae infection. WRKY33 functions as an activator and directly binds to the promoters of camalexin biosynthesis genes. Besides transcription factors, CALCIUM-DEPENDENT PROTEIN KINASE (CPK)5/6 and MAPK3/6 can phosphorylate WRKY33 to enhance promoter binding and transactivation. At the translational level, ribosome footprinting showed that genes involved in camalexin biosynthesis, CYP79B2 and CYP79B3, also increased translational efficiency under pattern triggered immunity. Despite the rich knowledge of these upstream regulators of the camalexin biosynthetic pathway, it remains unknown how the rapid induction upon a pathogen signal is enabled”.
In Discussion, we added: “Our results provide new evidence for how chemical defense mediated by camalexin may be regulated at the epigenetic level. However, we cannot rule out the possibility that other known mechanisms regulating camalexin genes may also affect the transcription kinetics and metabolite accumulation. For example, H3K27me3 affects gene expression by altering chromatin accessibility to transcription factors. Removing this repression mark may create a permissive environment and facilitate transcription factors, such as WRKY33, to bind to promoters of camalexin genes. At the translational level, camalexin biosynthesis genes can alter translational efficiency controlled by a highly enriched messenger RNA consensus sequence, R-motif, during pattern triggered immunity. Additional studies are needed to unravel how different regulatory machineries work together to enable the rapid induction of camalexin genes upon stress signals.”
Reviewer #2 (Public Review):
The authors propose that a bivalent chromatin switch exists on genes for the major Arabidopsis phytoalexin and this helps to influence the kinetics of this compounds regulation. This suggests that developmental regulatory designs are also used for defense chemistry. This is an interesting idea and the use of serial ChIP to show that this is not developmentally delineated but in fact occurring on a single promoter at the same time is very interesting. The efforts to extend this to being a general specialized metabolism pathways are less clear given some issues of over-counting pathways when a metabolic pathway is truly cyclical and applied to a hierarchical database design. The phenomon appears limited to fewer pathways then suggested and some statistical analysis are needed to support the claim on this one pathway.
Response: We appreciate hearing that our study that shows a bivalent chromatin regulating defense metabolism is interesting. Also, we appreciate the point about the apparent redundancy of reactions associated to pathways and how that may affect enrichment analysis. Metabolic pathways are interconnected, and metabolic genes can be mapped to multiple pathways. In addition, more than one variant of a pathway can be represented in a single species database. Finally, some pathways can be represented as a part of a super-pathway. The reviewer's comment prompted us to explore novel strategies to organize interconnected pathways without gene redundancy in our databases, which will be helpful to the community. But we feel that to do this analysis thoroughly and possibly overhaul the infrastructure of our database would be out of the scope of this manuscript. To address the comment about over-counting genes in the pathway enrichment analysis, we reported the number of specialized metabolic genes marked by both H3K27me3 and H3K18ac. We found that 37% genes (324 out of 887) annotated to specialized metabolism have both marks.
Regarding the comment about additional statistical analysis on the camalexin pathway, we analyzed the absolute transcript and metabolite levels using two-way ANOVA as the reviewer suggested.
Reviewer #3 (Public Review):
This study proposes the identification of "bivalent chromatin" at specialized metabolic gene clusters. Perturbation of either H3K27me3 or H3K18ac levels using mutants were used to show that there were effects on the expression of these metabolic genes. However, it is not novel that H3K27me3 and H3K18ac colocalize in the Arabidopsis genome. This was shown by Luo C et al in 2012 and is referenced by the authors. In fact, Luo C et al also showed these two modifications are colocalized by Chip-re-ChIP. This current study is presented as if the H3K18ac and H3K27me3 modifications are specific to metabolic genes, but they're not. Instead, it appears H3K18ac is localized to many H3K27me3 genes of which certain specialized metabolic genes are an enriched subset. There are >4000 genes targeted by PRC2/H3K27me3 in Arabidopsis that are enriched for genes that respond to the environment and or developmental cues. It is therefore, not unexpected that specialized metabolites are a subset of this class given they're only expressed in very specific environments. In summary, the results presented in Figure 1, 2 and 4 are essentially already published by Luo C et al, making the results of this study incremental.
Response: In our paper, we put forward a new model for the role of a bivalent chromatin formed by H3K18ac-H3K27me3, a form that is not yet studied in the bivalent chromatin literature, on regulating the timing of a defense compound synthesis pathway in a whole organism. As far as we know, bivalent chromatin has not been associated with controlling metabolism in any system. Furthermore, we tested our model genetically using several mutant lines that affect both the activating and repressing marks. By using a combination of epigenetic modification mutants, ChIP-re-ChIP, temporal transcriptomics and metabolomics on plants subjected to a pathogen signal, we showed that the bivalent chromatin is required to control the temporal gene expression kinetics of camalexin biosynthetic enzymes as well as the end product. This is the first time that a clear function of a bivalent chromatin has been demonstrated in vivo in any system. Moreover, we show that both H3K27me3 and H3K18ac are required to maintain timely induction of camalexin genes upon a stress signal, which is a novel molecular mechanism and different from PRC2-mediated repression.
The reviewer thought that we are claiming that H3K18ac and H3K27me3 are specific to metabolic genes. To avoid this confusion, we revised the manuscript to explicitly state that H3K27me3 as well as H3K18ac targets are enriched in specialized metabolism when compared to other domains of metabolism, but not exclusively targeting specialized or metabolic genes per se.
The paper the reviewer is referring to, Luo et al. (2012) The Plant Journal, is typical of the bivalent chromatin literature in that they observe certain patterns, but do not pursue it further. Luo et al. (2012) The Plant Journal reported genome-wide maps of nine histone modifications produced by ChIP-seq together with a strand-specific RNA-seq dataset to profile the epigenome and transcriptome in Arabidopsis thaliana. Combinatorial chromatin patterns were described by 42 major chromatin states with selected states validated using the re-ChIP assay. The major point of their paper is the potential synergistic effect of two repressive marks on natural antisense transcripts. However, because of the broad survey, they did find the H3K27me3-H3K4me3 bivalent chromatin. They also found a high correlation between H3K27me3 and H3K18ac, but do not go into any detail about this observation. They only description is: “Intriguingly, a strong correlation was detected between H3K18Ac and H3K27me3 in the Arabidopsis genome (Pearson r = 0.44, Figure 1b and Figure S4), although histone acetylation is not expected to co-localize with a repressive mark such as H3K27me3. The functional relevance of the co-existence of these two marks is unclear at this time.” (Luo et al. (2012) The Plant Journal).
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Evaluation Summary:
This study proposes the identification of "bivalent chromatin" in genes associated with the biosynthesis of secondary metabolites in Arabidopsis and describes an investigation into the role of chromatin states in the regulation of the major Arabidopsis phytoalexin. Perturbation of either H3K27me3 or H3K18ac levels using mutants were used to show that there were effects on the expression of these metabolic genes. It has previously been shown that H3K27me3 and H3K18ac colocalize in the Arabidopsis genome and that genes targeted by PRC2/H3K27me3 in Arabidopsis are enriched for genes that respond to the environment and/or developmental cues. Therefore, the reported changes to the regulation of these genes in defective mutants are as expected, although the finding of this study will still be of interest to those working …
Evaluation Summary:
This study proposes the identification of "bivalent chromatin" in genes associated with the biosynthesis of secondary metabolites in Arabidopsis and describes an investigation into the role of chromatin states in the regulation of the major Arabidopsis phytoalexin. Perturbation of either H3K27me3 or H3K18ac levels using mutants were used to show that there were effects on the expression of these metabolic genes. It has previously been shown that H3K27me3 and H3K18ac colocalize in the Arabidopsis genome and that genes targeted by PRC2/H3K27me3 in Arabidopsis are enriched for genes that respond to the environment and/or developmental cues. Therefore, the reported changes to the regulation of these genes in defective mutants are as expected, although the finding of this study will still be of interest to those working on pathogen-induced changes to plant metabolism.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The series of experiments to identify and examine changes in chromatin marks in response to flgg22 treatments investigate clearly defined hypotheses. They first present data showing that genes in pathways involved in specialized metabolism are more likely to be associated with both repression (H3K27me3) and activation (H3K18ac) marks than expected by chance in the genome. Using antibodies against H3K27me3 and H3K18ac in pull-down experiments, they show that H3K18ac and H3K27me3 are co-localized at the camalexin biosynthesis genes. They then show that, in response to FLG22, H3K18ac modifications increased and H3K27me3 modifications depleted. In mutant lines that have defective deposition of H3K27me3 expression of camalexin biosynthesis genes in response to FLG22 increased compared to wild type. Together, this …
Reviewer #1 (Public Review):
The series of experiments to identify and examine changes in chromatin marks in response to flgg22 treatments investigate clearly defined hypotheses. They first present data showing that genes in pathways involved in specialized metabolism are more likely to be associated with both repression (H3K27me3) and activation (H3K18ac) marks than expected by chance in the genome. Using antibodies against H3K27me3 and H3K18ac in pull-down experiments, they show that H3K18ac and H3K27me3 are co-localized at the camalexin biosynthesis genes. They then show that, in response to FLG22, H3K18ac modifications increased and H3K27me3 modifications depleted. In mutant lines that have defective deposition of H3K27me3 expression of camalexin biosynthesis genes in response to FLG22 increased compared to wild type. Together, this progression of experiments provides convincing data of an association between chromatin state and changes in transcription levels. Overall, the model is compelling, though the authors should take care to ensure that the language used is reflective of the association or interplay between chromatin state and transcription factor availability.
What is less clear is the link between chromatin states/transcriptional expression and the abundance of the metabolic pathway products that are required to limit pathogen spread. In this manuscript, Zhao et al, test camalexin content using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following FLG22, finding an initial accumulation, followed by further increases over 6 hours. Plants with reduced H3K27me3 marks started to accumulate camalexin earlier while those with reduced H3K18ac marks accumulated camalexin later. While the altered timings in the mutant lines do support a connection between gene expression dynamics and metabolite accumulation, it does not prove that changes in transcription are wholly responsible for the differences in metabolites. Previously reported Ribo-seq experiments mRNAs from genes, including those for camalexin biosynthesis, suggesting that PTI/RTI-triggered translational regulation has a significant role in changes in expression (Xu et al 2017 Nature 545, 487-490; Yoo et al Molecular Plant 13:1 88-98). This does not need to detract from the main findings of this paper, but the changes in metabolite accumulation should be interpreted with these data in mind and discussed appropriately.
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Reviewer #2 (Public Review):
The authors propose that a bivalent chromatin switch exists on genes for the major Arabidopsis phytoalexin and this helps to influence the kinetics of this compounds regulation. This suggests that developmental regulatory designs are also used for defense chemistry. This is an interesting idea and the use of serial ChIP to show that this is not developmentally delineated but in fact occurring on a single promoter at the same time is very interesting. The efforts to extend this to being a general specialized metabolism pathways are less clear given some issues of over-counting pathways when a metabolic pathway is truly cyclical and applied to a hierarchical database design. The phenomon appears limited to fewer pathways then suggested and some statistical analysis are needed to support the claim on this one …
Reviewer #2 (Public Review):
The authors propose that a bivalent chromatin switch exists on genes for the major Arabidopsis phytoalexin and this helps to influence the kinetics of this compounds regulation. This suggests that developmental regulatory designs are also used for defense chemistry. This is an interesting idea and the use of serial ChIP to show that this is not developmentally delineated but in fact occurring on a single promoter at the same time is very interesting. The efforts to extend this to being a general specialized metabolism pathways are less clear given some issues of over-counting pathways when a metabolic pathway is truly cyclical and applied to a hierarchical database design. The phenomon appears limited to fewer pathways then suggested and some statistical analysis are needed to support the claim on this one pathway.
-
Reviewer #3 (Public Review):
This study proposes the identification of "bivalent chromatin" at specialized metabolic gene clusters. Perturbation of either H3K27me3 or H3K18ac levels using mutants were used to show that there were effects on the expression of these metabolic genes. However, it is not novel that H3K27me3 and H3K18ac colocalize in the Arabidopsis genome. This was shown by Luo C et al in 2012 and is referenced by the authors. In fact, Luo C et al also showed these two modifications are colocalized by Chip-re-ChIP. This current study is presented as if the H3K18ac and H3K27me3 modifications are specific to metabolic genes, but they're not. Instead, it appears H3K18ac is localized to many H3K27me3 genes of which certain specialized metabolic genes are an enriched subset. There are >4000 genes targeted by PRC2/H3K27me3 in …
Reviewer #3 (Public Review):
This study proposes the identification of "bivalent chromatin" at specialized metabolic gene clusters. Perturbation of either H3K27me3 or H3K18ac levels using mutants were used to show that there were effects on the expression of these metabolic genes. However, it is not novel that H3K27me3 and H3K18ac colocalize in the Arabidopsis genome. This was shown by Luo C et al in 2012 and is referenced by the authors. In fact, Luo C et al also showed these two modifications are colocalized by Chip-re-ChIP. This current study is presented as if the H3K18ac and H3K27me3 modifications are specific to metabolic genes, but they're not. Instead, it appears H3K18ac is localized to many H3K27me3 genes of which certain specialized metabolic genes are an enriched subset. There are >4000 genes targeted by PRC2/H3K27me3 in Arabidopsis that are enriched for genes that respond to the environment and or developmental cues. It is therefore, not unexpected that specialized metabolites are a subset of this class given they're only expressed in very specific environments. In summary, the results presented in Figure 1, 2 and 4 are essentially already published by Luo C et al, making the results of this study incremental.
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